I. Field of the Invention
The present invention relates generally to the fields of antigens, antibodies and adjuvants. The invention particularly provides for the generation of enhanced immune responses by associating or incorporating an immunopotentiating agent onto a natural cellular membrane or into an intracellular compartment. In a specific instance, the invention is exemplified by incorporating an adjuvant, such as a lipopolysaccharide-like adjuvant, into tumor cells and onto their outer membranes.
II. Description of the Related Art
Methods for manipulating the immune system to achieve a desired effect have been known for many years, and are used both in the prevention and therapy of disease and in immunization protocols to generate specific antibodies for other uses, e.g., in diagnostics. However, generating an appropriate immune response is not always a straightforward matter. Particular problems arise with antigens that are "immunologically cryptic", in which cases the immune responses are often too weak to be of practical use.
The problems associated with generating immune responses apply to a wide range of clinical and laboratory protocols, with one of the most important areas being that of cancer treatment and therapy. Various modalities of therapy have been used during the past 30 years to treat cancer, including radiation and chemotherapy, radical surgery and immunologically-based protocols. The generation of immune responses to isolated tumor antigens using microbial adjuvants has also been described (U.S. Pat. No. 4,877,611). Melanoma cell vaccines using shed antigens are available (e.g., U.S. Pat. No. 5,194,384; Bystryn et al., 1988; Livingston et al., 1987a). However, this has certain limitations and melanoma still poses a significant health problem worldwide (Elder et al., 1995).
Portoukalian (1978) recognized the importance of gangliosides as tumor-associated antigens in human melanoma. Since this work, interest in the biochemical and immunological characteristics of tumor-related gangliosides has increased. Essentially, gangliosides are glycolipids containing sialic acids and are important membrane bound components of normal and neoplastic cells (Portoukalian, 1978). Immunologically, they are recognized as T-cell independent antigens (Hardings et al., 1991; Ishioka et al., 1992; Freimer et al., 1993) and suppressors of cellular immune functions (Kawaguchi et al., 1989; Morrison et al., 1989; Miller & Esselman, 1975; Lengle et al., 1979; Whisler & Yates, 1980; Prokazova et al., 1988; Portoukalian, 1989; Hoon et al., 1988; Chu & Sharom, 1993).
Solid tumors of neuroectodermal origin produce and shed large quantities of immunosuppressive sialoglycolipids or gangliosides (Ravindranath & Morton, 1991). Human cutaneous malignant melanoma expresses gangliosides GM.sub.3, GD.sub.3, GM.sub.2, GD.sub.2 and O-AcGD.sub.3 (Ravindranath & Irie, 1988), whereas B-16 murine melanoma expresses only GM.sub.3 (Takahashi et al., 1988). The shedding of gangliosides by solid tumors leads to significantly elevated levels of gangliosides in the sera of cancer patients, as compared to normal subjects (Kloppel et al., 1977; Horgan, 1982; Katopodis et al., 1982; Munjal et al., 1984; Dwivedi et al., 1990; Tautu et al., 1988).
Intraperitoneal administration of GM.sub.3 into the B16 melanoma-bearing mice has been reported to significantly augment melanoma growth, suggesting that GM.sub.3 shed from tumor cells may favor tumor growth, possibly by suppressing immune-surveillance (Takahashi et al., 1988). In support of this observation, it has also been demonstrated that GM.sub.3 preferentially suppresses the generation and activity of the cytotoxic lymphocytes in tumor bearing mice, suggesting that melanoma-derived GM.sub.3 in circulation may impede antitumor functions of the immune system. GM.sub.3 has also been found to be immunosuppressive in humans (Hachida et al., 1993; 1994).
Although anti-ganglioside antibodies are recognized as naturally occurring autoantibodies (Gillard et al., 1989), their level remains low even after repeated immunizations with purified gangliosides (Bogoch, 1960). Several investigators have attempted to induce antibody response to gangliosides by admixing gangliosides with foreign carrier proteins. These include, amongst others, pig serum (Rapport & Graf, 1969; Sherwin et al., 1964); serum albumin (Pascal et al., 1966; Koscielak et al., 1968); human erythrocyte glycoprotein (Naiki et al., 1974); foreign erythrocytes (Yokoyama et al., 1963); and a mixture of meningococcal outer membrane proteins, cationized bovine serum albumin, multiple antigenic peptides, polylysine and keyhole limpet (molluscan) hemocyanin (Helling et al., 1993).
The use of non-toxic microbial adjuvants with isolated and purified tumor-associated antigens was described in U.S. Pat. No. 4,877,611. The use of bacterial carriers, such as Salmonella Minnesota or Mycobacterium bovis (Livingston et al., 1987a; U.S. Pat. No. 5,102,663) has also been reported to augment antibody response against gangliosides in human and in murine studies. However, GM.sub.3 bound to cell membranes in humans (Livingston et al., 1987b), and purified and free GM3 in mice (Livingston et al., 1987a), induced poor antibody responses. It has not been established whether the failure to induce antibody responses is due to crypticity of the antigen or failure to be recognized by the antigen presenting system.
It has been shown that attaching lipopolysaccharide (LPS) and lipid A to synthetic membranes (liposomes) can result in the generation of an immune response to membrane components. This has been proposed to be connected with macrophage recruitment (Verma et al., 1992). Research has shown that immunization of mice with lipid A-attached liposomes induced antibody against components of the liposomes, whereas liposomes without lipid A failed to elicit any response (Schuster et al., 1979; Banerji et al., 1982; Verma et al., 1992). Recently, Freimer et al. (1993) have also studied the T-cell independent antibody response to purified gangliosides using lipid A as an adjuvant. However, the use of lipid A as an immunological adjuvant in humans is precluded by the toxicity of lipid A.
Monophosphoryl lipid A (MPL) is a nontoxic derivative of lipid A from Salmonella (Qureshi et al., 1985; Ribi, 1984; Johnson et al., 1987). MPL has comparable biological activities to lipid A, including B cell mitogenicity, adjuvanticity, activation of macrophages and induction of interferon synthesis (Ribi et al., 1984, 1986; Verma et al., 1992). Johnston & Bystryn (1991) tested the combined effect of mycobacterial cell wall skeleton and MPL on a melanoma vaccine of murine B16 cells. Unfortunately, MPL was not found to potentiate tumor-protective immunity in these studies (Johnston & Bystryn, 1991).
Livingston et al. (1987a) have tested the usefulness of MPL in augmenting anti-ganglioside antibodies, using purified gangliosides and ganglioside-liposomes. However, the Livingston et al. group prefer to use whole Salmonella as an adjuvant, as shown in their subsequent studies in U.S. Pat. No. 5,102,663. In U.S. Pat. No. 5,312,620, a complex system of polymeric adjuvants incorporated into lipid layers is described. The adjuvants are first conjugated to a polymerizable group and then co-polymerized with a water-soluble and/or amphiphilic polymerizable monomer or combined with a polymerized amphiphile.
Despite the continuing efforts in this field, it is apparent that improved methods and novel strategies for generating immune responses are still needed. Simple methods that are appropriate for use with a wide variety of antigens are particularly desirable. The development of a method by which to improve the immune response against immunologically cryptic antigens would represent a significant advance, particularly if such a method was adaptable for use against clinically relevant antigens.